The development of an accurate gene map is a primary goal of cytogenetic studies in any organism. Chromosomal, band and sub-band localization of genes in animal genomes can elucidate the evolution of genome organization. Moreover, knowledge of genome organization facilitates integration with human medical research of genetic studies of animal models such as canine behavioral traits and genetic diseases. However, the karyotype of the 78 dog chromosomes is incompletely standardized, and to date no autosomal localization by FISH of a canine gene or anonymous sequence has been reported. We analyzed pre-harvest treatment with EtBr and/or BrdU for dog lymphocyte chromosomes. High quality metaphases for FISH were obtained with BrdU, and this treatment also allowed for simultaneous fluorescent banding under UV light with FISH. Conditions for FISH were optimized using both canine genomic probes (phage and plasmids) and cross-species hybridization with human YACs. The X-linked genes F8 and F9, HPRT, XIST, PGK and CHM, and the MHC locus (DLA) and the immunoglobulin heavy chain locus have been mapped in the dog by FISH. Interestingly, not only the X chromosomal assignment, but also the order and band localization of all genes investigated to date are identical in canine and human X chromosomes. Further studies are focused on inclusion of cat chromosomes, more tractable for karyotypic analysis, and mapping of the locus for canine narcolepsy, which has been extensively studied and occurs as a simple dominant trait in large pedigrees of dogs studied by our Stanford University Medical Center collaborator, Emmanuel Mignot. Reduction of the background signal produced by yeast DNA present in human YAC clone probes will be a focus of ongoing protocols, as this is a major impediment to using human YACs as cross-species probes for physical mapping.